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K Morihara

Publications and source records attributed to K Morihara.

17 recordsLinked to original sources
PubMed1992

Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase.

The DNA encoding the collagenase of Vibrio alginolyticus was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited both collagenase antigen and collagenase activity. The open reading frame from the ATG initiation codon was 2442 bp in length for the collagenase structural gene. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature collagenase consists of 739 amino acids with an Mr of 81875. The amino acid sequences of 20 polypeptide fragments were completely identical with the deduced amino acid sequences of the collagenase gene. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified collagenase reported previously. The analyses of both the DNA and amino acid sequences of the collagenase gene were rigorously performed, but we could not detect any significant sequence similarity to other collagenases.

Amino Acid Sequence↗
PubMed1979

Protease and elastase of Pseudomonas aeruginosa: inactivation of human plasma alpha 1-proteinase inhibitor.

The present study indicates that crystalline elastase of Pseudomonas aeruginosa is a very potent inactivator of human plasma alpha 1-proteinase inhibitor, the enzyme (E) inactivated the inhibitor (I) almost completely within 1 h at 25 degrees C at a molar ratio of E/I = 1:100. The crystalline P. aeruginosa protease also inactivated the inhibitor, but 100-fold less. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha 1-proteinase inhibitor inactivated by the elastase and protease showed decreases in molecular weight of approximately 5,000 and 10,000, respectively. Regeneration of trypsin was negligible even when bovine trypsin-alpha 1-proteinase inhibitor complex (E/I = 1.0) was treated with the elastase. The affinity of alpha 1-proteinase inhibitor to trypsin was much higher than that to elastase. It was suggested that, assuming the pseudomonal proteases are produced and can inactivate alpha 1-proteinase inhibitor in vivo during pseudomonal diseases, the loss of alpha 1-proteinase inhibitor activity may permit the endogenous serine proteases to cause tissue destruction.

Humans↗
PubMed1979

Protection against hemorrhagic pneumonia of mink by Pseudomonas aeruginosa multicomponent vaccine.

An attempt to prevent epidemics of hemorrhagic pneumonia in mink due to Pseudomonas aeruginosa was made in the course of epidemics with injection of the multicomponent vaccine consisting of common protective antigen (OEP) of P. aeruginosa mixed with toxoids of protease and elastase of the bacillus. Enzootics of hemorrhagic pneumonia, due to P. aeruginosa serotype 8, broke out from August to October 1977 in a total of 13 sheds of 3 farms (A, B and C) which were located in the northeast area of Hokkaido. These farms were raising 7,452, 2,553 and 10,639 mink respectively. The mortality rate of the mink on farms A, B and C were 11.8%, 13.0% and 1.0% respectively. The vaccination was performed on the 3 farms 5, 8 and 21 days after the onset of the disease. Inoculation of each mink with 200 micrograms or 100 micrograms of each of the three components of the multicomponent vaccine was effective in most of the male and female groups of mink. The period required for revealing the effect of the vaccination was very short, in some cases only a few days. Administration of the vaccine 21 days after the onset of the enzootic was also effective.

Animals↗
PubMed1979

Pepstatin-insenstive acid proteases from Scytalidium lignicolum. Kinetic study with synthetic peptides.

A kinetic study was conducted on the acid proteases A-1 and A-2 from Scytalidium lignicolum using synthetic peptides as substrates. Almost maximum activity was attained with N-acylated tetrapeptides as the molecular size of substrates was increased. Suitable amino acid residues were required at the P1-P2 and P1'-P2' positions [notation of Schechter and Berger (14)]. Hydrophobic or bulky residues such as leucine were specifically required at the P1 and P1' positions, with the specificity at the latter position being considerably lower than that at the former. For catalysis, the presence of certain amino acid residues at the P2 and P2' positions was essential, mainly in relation to kcat. An inhibition study supported this view. Stringent stereospecificity was observed at the P2 and P2' positions, but the side chain specificity was low. Study of the B enzyme from the same organism was very difficult owing to its low activity against the peptides used. The Scytalidium acid proteases A-1, A-2, and B showed considerably different behavior against peptide substrates in comparison with usual acid proteases, which are senstive to pepstatin.

Aspergillus niger↗
PubMed1978

Peptide bond synthesis catalyzed by alpha-chymotrypsin.

alpha-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of alpha-chymotrypsin and starting materials were required for synthesis. Four amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal position. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.

Amines↗
PubMed1978

Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), protease and elastase toxoids of Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink due to P. aeruginosa.

Toxoids of protease and elastase of Pseudomonas aeruginosa were successfully prepared by treatment with 8% formalin plus 0.2 m lysine and by 4% formalin respectively. The two toxoids proved sufficiently potent to elicit high antibody titers as estimated by both the enzyme-neutralizing and passive hemagglutination tests. The effectiveness of immunizing minks with a single component-vaccine consisting of the common antigen (OEP) of P. aeruginosa or the protease toxoid (PT) or the elastase toxoid (ET) and with the two (PT and ET) or the three (OEP, PT and ET) component-mixed vaccines, on hemorrhagic pneumonia in minks due to the bacteria was investigated. Female Sapphire minks, 3.5 months old, were used in two experiments performed in 1975 and 1976. Minks were immunized three times in one month with a total of 1 mg of each of the three antigens in the case of a single component vaccine and with a total of 2 or 3 mg (equal amounts of each component) in the case of the two or three component vaccines. Two or three weeks after the last immunization, challenge exposure with strain No. 5 was carried out by intranasally inoculating an inoculum containing serial dilutions of 10(3) -10(10) of live bacteria. Summarizing the results of the two experiments, in the case of controls, nonimmunized minks and minks immunized with potassium aluminum sulfate (potash alum) alone, the LD50 values were approximately 10(3) -10(4) with no significant difference between the two. In the case of OEP-vaccinated minks, the LD50 value was about 10(6) and thus clearly differed from those of the controls. In minks immunized with the three-component-vaccine, however, the LD50 value was about 10(8) -10(9), which indicated that the three-component-mixed vaccine was remarkably more effective than the single OEP vaccine component. In minks immunized with either PT or ET or both, the LD50 values were about 10(8) -10(9). The effectiveness of the vaccine made with ET or PT alone is discussed in the text. The pathological findings of the minks which died or survived are described.

Animals↗
PubMed1977

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.

Amino Acids↗
PubMed1977

Trypsin as a catalyst for peptide synthesis.

Trypsin-catalyzed syntheses of peptides were performed using various N-acylated amino acid or peptide esters as donors and amino acid derivatives, peptides, or their derivatives as acceptors. The synthesis was almost quantitative under optimal conditions. Considerably more enzyme and a more alkaline pH were necessary for synthesis than hydrolysis. Another very important condition was the concentration of the starting materials; higher concentrations resulted in much better product yields. The nucleophile specificity for synthesis was also important; hydrophobic or bulky amino acid residues were most efficient at the P1' position, and L-proline as well as D-amino acid residues were the worst choices. The synthesis was also dependent on the solubility of the products synthesized; the yield was higher with products of lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible using N-acylated peptide esters and various peptides. The present study suggests that trypsin may become a useful tool for peptide synthesis.

Animals↗
PubMed1977

Production of protease and elastase by Pseudomonas aeruginosa strains isolated from patients.

Using 20 strains of Pseudomonas aeruginosa isolated from patients, production of protease, elastase, and collagenase was determined by shaking culture in either complex or semisynthetic medium. No collagenase was produced by any strain of P. aeruginosa. According to their production of protease and elastase in different media, the P. aeruginosa strains were divided into three groups: the first group can produce elastase in complex medium and both protease and elastase in semisynthetic medium; the second group cannot produce any proteolytic enzymes in complex medium but can produce any proteolytic enzymes in either medium; and the third group cannot produce any proteolytic enzymes in either medium. In spite of the differences in the ability of the strains to produce the enzymes, depending upon the origin of the strains, the protease or elastases produced in different broths were regarded as identical.

Caseins↗
PubMed1975

Effects of protease and elastase from Pseudomonas aeruginosa on skin.

Protease and elastase were inoculated intracutaneously in rabbits, the process of which was followed. Of the enzymes, 50 mug caused circumscribed hemorrhage at the inoculated local and subcutaneous part, and ulcerating lesions of skin and extensive hemorrhagic lesion at the subcutaneous tissues were observed in the case of inoculation with 250 mug. The doses amounting to 500 to 1000 mug of the enzymes caused extensively ulcerating, necrotic lesions at the skin and extensive hemorrhage at the subcutaneous tissue, and a large amount of hemorrhage was observed in the abdomen. It was observed microscopically that cellular infiltration and hemorrhage in the subcutaneous tissue and muscular layer were caused by administration of 2 to 10 mug of the enzymes, and that degeneration of endothelial cells was caused by 2 mug of them.

Animals↗
PubMed1975

In vivo studies on protease and elastase from Pseudomonas aeruginosa.

Protease and elastase from Pseudomonas aeruginosa were inoculated in female mice by intravenous, intraperitoneal, intrapleural and intranasal route, and the lethality and damage of various organs were examined. The protease and elastase exhibited respectively the following minimum lethal dose (MLD) values in 24 hr; 300 and 375 mug inoculated intravenously; 200 and 125-250 mug intraperitoneally; and 100 and 62.5 mug intrapleurally. The instillation of a defined dose of the enzyme by the intranasal route was difficult to control, therefore the MLD could not be defined exactly. The protease and elastase were capable of eliciting hemorrhage at various organs of mice according to the route of inoculation. Of protease, intravenous injections elicited petechial hemorrhage at the lungs and parietal bone-area, and severe one in the medullary area of kidney. The intraperitoneal injection resulted in petechial hemorrhage of the lungs, diaphragm, peritoneum and gastrointestinal serosa. Intrapleural injections and intranasal instillation resulted in confluent pulmonary hemorrhage. Of elastase, intravenous injections elicited confluent pulmonary hemorrhage, hemorrhage in the medullary area of kidney and cerebral ventricles, and petechial hemorrhage at the stomach-walls. The intraperitoneal injections resulted in petechial hemorrhage at the lungs, diaphragm, peritoneum and gastointestinal serosa. Intrapleural injections resulted in confluent pulmonary hemorrhage, and petechiae at the diaphragm and pleura. Intranasal instillation resulted in confluent pulmonary hemorrhage.

Animals↗